准噶尔盆地大沙鼠鼠疫自然疫源地鼠疫耶尔森菌质粒谱的研究

目的 了解和掌握准噶尔瓮地大沙鼠鼠疫自然疫源地鼠疫菌质粒谱类型.方法 提取新疆地区不同鼠疫自然疫源地及青藏高原喜玛拉雅旱獭鼠疫自然疫源地、昆仑山喜马拉雅旱獭鼠疫自然疫源地和内蒙古长爪沙鼠鼠疫自然疫源地的39株鼠疫菌质粒DNA,采用琼脂糖凝胶电泳法对其进行质粒谱图型分析.结果 在检测的26株准噶尔盆地大沙鼠鼠疫自然疫源地鼠疫菌中,有23株质粒谱为6×106、45×106、65×106,3株为6×106、45×106和72×106.结论 准噶尔盆地大沙鼠鼠疫自然疫源地鼠疫菌质粒谱分为两个类型:一为6×106、45×106、65×106质粒谱型,是该疫源地的主要质粒谱型,与邻近的北天山灰旱獭.长尾黄鼠鼠疫自然疫源地及内蒙占长爪沙鼠鼠疫自然疫源地、昆仑山喜马拉雅旱獭鼠疫自然疫源地和青藏高原喜马拉雅旱獭鼠疫自然疫源地的鼠疫菌主要质粒谱型一致;二为6×106、45×106和72×106质粒谱型,为少数型,在我国系首次发现.     

应用抑制削减杂交技术进行鼠疫耶尔森菌的比较基因组研究

目的确定古典型鼠疫耶尔森菌的特有序列，为建立和完善该菌基因组分型系统提供可靠数据。方法通过抑制削减杂交技术发现差异片段并应用PCR验证。结果发现了5个在不同生物型鼠疫菌问存在差异的DNA片段，其中一个383bp的片段在来自天山山地灰旱獭、长尾黄鼠鼠疫自然疫源地的30株鼠疫菌全部阳性，而来自其它鼠疫自然疫源地的菌株全部阴性，5株假结核耶尔森菌中有2株(生物Ⅰ型和Ⅱ型)阳性。结论该383bp片段为天山山地灰旱獭、长尾黄鼠鼠疫自然疫源地的鼠疫菌所特有，此疫源地的菌株可能是我国较古老的鼠疫菌。     

天山山地灰旱獭-长尾黄鼠鼠疫疫源地鼠疫菌基因分型研究

目的对天山山地灰旱獭．长尾黄鼠鼠疫疫源地鼠疫菌进行基因分型。方法根据已经证实的22个差异区段（DFR）设计引物，对每株鼠疫菌进行PCR扩增。结果61株天山疫源地鼠疫菌株共包括4个基因型，即1、2、3、4型。基因型1、2和3在北天山疫源地西部鼠疫菌株中都有分布，但尼勒克菌株的基因型全部为1型。南天山疫源地阿合奇和阿图什两个县菌株的基因型存在显著差别，阿合奇菌株基因型与北天山疫源地的菌株比较接近，而3株阿图什菌株的基因型均为4型，与帕米尔高原长尾旱獭鼠疫疫源地鼠疫菌株相同。结论基因分型结果与纪树立的生态分型基本一致。天山疫源地和帕米尔高原疫源地在阿图什县有交叉。     

我国鼠疫菌毒力因子的比较与分析

目的 研究自不同生态型和不同鼠疫自然疫源地分离的鼠疫菌4种毒力因子的性状特征。方法 常璺方法检查鼠疫菌毒力因子性状并比较在不同疫源地之间、不同生态型之间的差异。结果 1326株鼠疫菌中仅1株缺失FI、6株缺失PstI,Pgm和WW阳性分别为92.99%、76.47%。结论 自我国不同疫源地、平淡同生态型和不同年代分离的野生型鼠疫菌绝大多数能产生FI和PstI,且不同生态型和不同疫源地分离的菌株这两种毒力因子无明显差别,性状稳定,VW^-和Pgm^-菌株与生态型及疫源地有一定的关系。经长期人工培养传代后。4种毒力因子相比,VW因子较易丧失,该毒力因子具有更不稳定特点。     

新疆山地鼠疫自然疫源地鼠疫耶尔森菌基因组分化和演变

目的比较来自我国新疆4大片山地鼠疫自然疫源地的144株鼠疫菌的基因组组成的差别,研究新疆山地鼠疫自然疫源地鼠疫菌的基因分型和进化规律。方法根据已经证实的22个差异区段(DFR)设计引物,每株鼠疫菌的每个DFR都采用PCR技术进行验证。结果新疆4大片山地鼠疫自然疫源地的144株鼠疫菌的基因型DFR图谱可归类为14种,分为7个主要基因组型和7个次要基因组型。西天山北坡灰旱獭-长尾黄鼠鼠疫疫源地存在3个主要基因组型和5个次要基因组型,西段以01型基因组型为主,占种群体的91.7%,02型占8.3%;中段以03型为主,占68.8%,01型占4.2%,02型占20.85%,另有02M1和02M3(02型的突变型)占2.1%,03M1型(03型的突变型)占2.1%;东段以02型为主,89.8%,01型、02M2、02M3和02M4型(02型的突变型)各占2.0%。南天山灰旱獭鼠疫疫源地存在2个主要基因型和1个次要基因型,以4型为主,占66.7%,02型占16.7%, 04M1型(04型的突变型)占16.7%。帕米尔高原-阿赖山红旱獭鼠疫疫源地仅存在04型1种基因型。昆仑山喜马拉雅旱獭鼠疫疫源地分为中昆仑和东昆仑鼠疫自然疫源地,中昆仑山存在2种鼠疫基因组型,以11型为主,占群体的90%,12型占10%;东昆仑山存在3种鼠疫基因组型,以05型为主,占66.7%,02型和11型分别占16.7%。结论新疆山地鼠疫自然疫源地鼠疫菌基因组的演化由3个演化路线组成。西天山北坡鼠疫自然疫源地的鼠疫菌基因组,自西向东在不同的生态地理环境和宿主媒介作用下发生适应性进化和演变,由较为古老的01型基因组逐渐演化为02和03型,最后南下至青海和东昆仑演变为05型,并且在这条演化路线上存在许多演变过程中发生的基因组地域交叉和次要基因组型;南天山和帕米尔高原-阿赖山鼠疫自然疫源地的鼠疫菌的04型基因组型是由01型直接演化而来,并在这一区域形成主要鼠疫基因组型;中昆仑山鼠疫自然疫源地的鼠疫菌基因组型可能是由西藏冈底斯山喜马拉雅旱獭鼠疫源地的鼠疫菌的10型基因组演化而来,形成以11型为主,12型为辅的鼠疫基因组构型特点。     

44株中国田鼠型与24株非田鼠型鼠疫菌的DFR检测分析

目的比较我国两个田鼠鼠疫自然疫源地的44株田鼠型鼠疫菌的基因组组成，研究中国田鼠型鼠疫菌的遗传稳定性。方法根据已经证实的22个差异区段(I)FR)设计引物，每株鼠疫菌的每个DFR都采用PCR技术进行验证。结果22个DFR在两种田鼠型鼠疫菌的分布状态完全一致，即均同时缺失了DFR6、DFR11、DFR12、DFR13、DFR18、DFR19和DFR20，与来源于其他鼠疫自然疫源地非田鼠型鼠疫菌的基因组成有显著差别。结论锡林郭勒高原型鼠疫菌和青藏高原青海田鼠型鼠疫菌在基因组组成上高度一致，田鼠型鼠疫菌的基因缺失可能与其对人不致病有关。     

云南、福建和贵州省鼠疫耶尔森菌基因分型研究

云南省位于我国西南边陲，存在滇西山地齐氏姬鼠大绒鼠鼠疫自然疫源地（野鼠鼠疫疫源地）和滇西山地闽广沿海居民区黄胸鼠鼠疫自然疫源地（家鼠鼠疫疫源地），除了剑川地区属于野鼠鼠疫疫源地外，云南省的西北部疫区均属于家鼠鼠疫疫源地。福建省位于我国东南沿海，其境内的鼠疫自然疫源地也属于家鼠鼠疫疫源地。贵州省兴义市位于云南和广西省交界处，也属于家鼠鼠疫疫源地。为了比较家鼠及野鼠疫源地鼠疫耶尔森菌（以下简称鼠疫菌）基因组成的差别，采用PCR技术对分离自三省的69株鼠疫菌进行了基因分型研究。     

准噶尔盆地鼠疫耶尔森菌的基因组型分析

目的 对我国新发现的准噶尔盆地大沙鼠鼠疫自然疫源地的鼠疫菌株进行基因组差异性区段的比较分析,确定该鼠疫疫源地鼠疫菌株的基因组类型.方法 按常规方法提取鼠疫菌染色体DNA,依据已证实的鼠疫菌22个差异性区段(DFR)设计引物,采用PCR技术检测鼠疫菌基因组差异性基因片段的构成.结果 准噶尔盆地鼠疫菌株的差异性区段结构类型为缺失DFRO1、04、06、07、10、13和DFR15～18,不同于其他鼠疫疫源地鼠疫菌株的结构类型.为我国新的鼠疫菌基因组型.结论 准噶尔盆地大沙鼠鼠疫疫源地鼠疫菌株的基因组结构类型为我国新的基因组类型.     

松辽平原达乌尔黄鼠鼠疫自然疫源地鼠疫茵基因分型

为了研究松辽平原达乌尔黄鼠鼠疫自然疫源地鼠疫耶尔森菌（以下简称鼠疫菌）的基因组成，采用PCR技术对分离自该疫源地88株鼠疫菌进行了基因分型，其中黑龙江4株、吉林43株、辽宁4株、内蒙37株，鼠疫菌DNA均由青海省地方病预防控制所鼠疫菌专业实验室提供。     

甘宁黄土高原阿拉善黄鼠鼠疫疫源地鼠疫菌的基因分型研究

目的 研究我国甘宁黄土高原阿拉善黄鼠鼠疫疫源地鼠疫菌的基因分型。方法 根据已经证实的22个DFR（差异区段）设计引物，每株鼠疫菌的每个DFR都采用PCR技术进行验证。结果 17株甘宁黄土高原疫源地鼠疫菌株共包括3个基因型，即7、11和13型。7、11和13型所占比例分别为5．9％（1／17）、5．9％（1／17）和88．2％（15／17）。结论 我国甘宁黄土高原阿拉善黄鼠鼠疫疫源地鼠疫菌的基因型主要为13型，而且13型是该疫源地鼠疫菌所特有的基因型。     

Human plague in the southwestern United States, 1957-2004: spatial models of elevated risk of human exposure to Yersinia pestis

Plague is a rare but highly virulent flea-borne zoonotic disease caused by the Gram-negative bacterium Yersinia pestis Yersin. Identifying areas at high risk of human exposure to the etiological agent of plague could provide a useful tool for targeting limited public health resources and reduce the likelihood of misdiagnosis by raising awareness of the disease. We created logistic regression models to identify landscape features associated with areas where humans have acquired plague from 1957 to 2004 in the four-corners region of the United States (Arizona, Colorado, New Mexico, and Utah), and we extrapolated those models within a geographical information system to predict where plague cases are likely to occur within the southwestern United States disease focus. The probability of an area being classified as high-risk plague habitat increased with elevation up to approximately 2300 m and declined as elevation increased thereafter, and declined with distance from key habitat types (e.g., southern Rocky Mountain pi?on--juniper [Pinus edulis Engelm. and Juniperus spp.], Colorado plateau pi?on--juniper woodland, Rocky Mountain ponderosa pine (Pinus ponderosa P.& C. Lawson var. scopulorum), and southern Rocky Mountain juniper woodland and savanna). The overall accuracy of the model was >82%. Our most conservative model predicted that 14.4% of the four-corners region represented a high risk of peridomestic exposure to Y. pestis.     

Virulence markers of LCR plasmid in Indian isolates of Yersinia pestis

Presence of 10 important yop genes in Yersinia pestis isolates (18 in number) of Indian origin from 1994 plague outbreak regions of Maharashtra (6 Rattus rattus & Tetera indica rodents) and Gujarat (11 from human patients, 1 from R. rattus) and from plague endemic regions of the Deccan plateau (8 from T. indica) was located by PCR and specific enzyme immunoassay. PCRs were standardized for six effector yops (YopE, YopH, YopJ, YopM, YopO and YopT), three translocator yops (YopB, YopD and YopK) and a regulator LcrV gene. Amplification of all the 10 yop genes was observed in isolates recovered from pneumonic patients and in 5 of 7 rodents from outbreak regions. Among these, amplification of the yopD gene was absent in all eight isolates, and that of yopM in all except one (10R). One of the isolates from rodents of the Deccan plateau (24H) was consistently negative for all the yops. Cloning and expression of truncated yopM (780 bp), yopB (700 bp) and lcrV (796 bp) genes in pQE vectors with SG13009 host cells yielded recombinant proteins for generation of monoclonal antibodies for further use in enzyme immunoassay. Ten stable reactive clones for YopB, nine for YopM and six for LcrV were obtained, all of them exhibiting specific reactions only to Y. pestis. Testing of 26 Y. pestis isolates by monoclonal antibody dot-ELISA and Western blotting provided results identical to PCR, suggesting that the isolates that failed to show PCR amplification also had no expression of their respective proteins. The Y. pestis isolates of outbreak regions had their virulence factors intact in the LCR plasmid. Yersinia pestis isolates recovered from rodents of the Deccan plateau were relatively heterogeneous. It appears that a long residency of Y. pestis of nearly 100 years in the enzootic plague foci has resulted in shedding of virulence genes in the LCR plasmid region in a fairly large proportion of the organisms, possibly due to natural recombination.     

Polymorphisms in the lcrV gene of Yersinia enterocolitica and their effect on plague protective immunity

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

Immune response to plasmid- and chromosome-encoded Yersinia antigens.

The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of yersinia pestis and Yersinia enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica strains with and without the virulence plasmids pYV019 and pYV8081, respectively, were resolved by denaturing electrophoresis, and the major antigens were visualized with sera from convalescing plague patients, individuals immunized with plague vaccine, and mice and rabbits immunized with avirulent live yersiniae. The Y. pestis grown in vitro in this study did not express detectable amounts of plasmid-encoded antigens. The sera from plague patients recognized Y. pestis and Y. enterocolitica antigens ranging from 15 to 72 kilodaltons (kDa), whereas sera from immunized subjects recognized four antigenic components in Y. pestis ranging from 17 to 64 kDa and five antigens in Y. enterocolitica ranging from 16 to 68 kDa. Sera from mice reacted with 7 antigens in Y. pestis and 12 antigens in Y. enterocolitica ranging from 14 to 68 kDa, and sera from rabbits reacted with 7 and 10 antigens in Y. pestis and Y. enterocolitica, respectively. All of the plague patient sera, as well as the sera from immunized mice and rabbits, reacted with a 22-kDa Y. enterocolitica plasmid-associated polypeptide, and five of the patient sera also recognized a Y. enterocolitica plasmid-associated 31-kDa protein. The results indicate a common immune response to at least these two plasmid-specified Yersinia outer membrane proteins. Y. pestis apparently expresses these components only in vivo, and in vitro, Y. enterocolitica expresses a greater number of plasmid-associated antigens than does Y. pestis.     

Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague

The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.